35 research outputs found
Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies
<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys<sup>448 </sup>with Cys<sup>483 </sup>stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p
Synergistic induction of lipid catabolism and anti-inflammatory lipids in white fat of dietary obese mice in response to calorie restriction and n-3 fatty acids
Employment Engagement
Employee engagement has been defined in many
different ways. Most often it has been defined as
emotional and intellectual commitment to the
organization (Baumruk 2004; Richman 2006;
Shaw 2005) or the amount of discretionary effort
exhibited by employees in their jobs (Frank et al.
2004).
In the academic literature, a number of definitions have been provided. Kahn (1990, p. 694)
defines personal engagement as “the harnessing of
organization members’ selves to their work roles;
in engagement, people employ and express themselves physically, cognitively, and emotionally
during role performances.” Thus, engagement
means to be psychologically present when occupying and performing an organizational role.
It has been also defined as psychological presence but he goes further to state that it involves
two critical components: attention and absorption.
Schaufeli et al. (2002) define engagement “as a
positive, fulfilling, work-related state of mind that
is characterized by vigor, dedication, and absorption.” They further state that engagement is not a
momentary and specific state, but rather it is “a
more persistent and pervasive affective-cognitive
state that is not focused on any particular object,
event, individual, or behavior.